Investigation of novel pathways causing autoinflammatory disease
Investigation of novel pathways causing autoinflammatory disease
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DissertationPrüfungsdatum
12.04.2022Datum der Veröffentlichung
17.05.2022Erstgutachter
Geyer, MatthiasZweitgutachter
Masters, SethMetadaten
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Inhalt
Autoinflammatory diseases (AIDs) are rare pathologies caused by mutations in genes associated with innate immune pathways. Investigating the underlying molecular defects contributes to identification of targeted treatment strategies for patients and steadily extends our knowledge of innate signalling pathways and their regulation. Two different diseases were the focus of this thesis: COPA syndrome and NLRC4-AID.
COPA syndrome is an autosomal dominantly inherited immune disorder, caused by loss of function mutations in the coat protein complex I (COPI) subunit α (COPA), which participates in retrograde vesicular trafficking of proteins from the Golgi apparatus to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency of COPA is associated with ER stress, NF-κB activation and type I interferon (IFNαβ) signalling, although the upstream innate immune sensor that is responsible for this was unknown.
Using a range of biochemical and molecular biological techniques, we established in vitro models that recapitulate unprovoked proinflammatory signalling upon CRISPR/Cas9-mediated deletion of COPA and identify aberrant activation of immune sensor STING, which was also dependent on upstream cytosolic DNA sensor cyclic GMP-AMP-synthase (cGAS).
Further, we demonstrate that genetic deletion of other COPI subunits, COPG1 and COPD, similarly induces STING activation, which suggests that innate immune diseases associated with mutations in other COPI subunit genes may exist.
Finally in this chapter, we show that inflammation in COPA syndrome patient PBMCs and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS-STING pathway as a novel therapeutic approach to treat inflammation associated with COPA syndrome and perhaps other COPI-deficiencies.
In the second and third part of this thesis, we focussed on NLRC4-associated AIDs, which are typically caused by autosomal dominant gain of function mutations in NLR family CARD domain containing 4 (NLRC4) that drive hyperactive inflammasome signalling.
Here, we report the first case of a patient with immune dysregulation carrying a recessive mutation in NLRC4 (c.478G>A, p.A160T). Using historical clinical data, serum cytokine analysis and in vitro stimulation of monocyte-derived macrophages we suggest aberrant inflammasome signalling as underlying cause of the inflammatory episodes experienced by the patient.
In vitro, we assessed the pathogenic potential of this mutation and demonstrate ligand-induced increased ASC specking, pyroptosis and cytokine secretion by NLRC4 A160T. Further, using meta-analysis association data we find that heterozygous allele carriers in the general population are at increased risk of developing ulcerative colitis (UC).
Results from subsequently performed structural and biophysical studies aiming to elucidate the conformational changes associated with this mutation point towards a mechanism of action that only affects NLRC4 A160T in the active conformation. Furthermore, since residue A160 is conserved in mice and most primate NLRC4 proteins, but T160 is encoded in the wildtype sequence of NLRC4 in ungulate and carnivore species, the evolutionary relationship between these species is presented. Finally, we discuss potential mechanisms of actions by which NLRC4 A160T may cause aberrant inflammasome activation and evaluate residue T160 as a potential phosphorylation site.
Together, studies described in this thesis identify a direct target for therapeutic intervention in immune diseases associated with defective COPI transport and shape the current understanding of retrograde trafficking as negative regulatory mechanism in STING signalling. Further, we provide in vitro evidence for the pathogenic potential of the NLRC4 A160T mutation, which was previously unrecognized and may represent a susceptibility locus for UC.
COPA syndrome is an autosomal dominantly inherited immune disorder, caused by loss of function mutations in the coat protein complex I (COPI) subunit α (COPA), which participates in retrograde vesicular trafficking of proteins from the Golgi apparatus to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency of COPA is associated with ER stress, NF-κB activation and type I interferon (IFNαβ) signalling, although the upstream innate immune sensor that is responsible for this was unknown.
Using a range of biochemical and molecular biological techniques, we established in vitro models that recapitulate unprovoked proinflammatory signalling upon CRISPR/Cas9-mediated deletion of COPA and identify aberrant activation of immune sensor STING, which was also dependent on upstream cytosolic DNA sensor cyclic GMP-AMP-synthase (cGAS).
Further, we demonstrate that genetic deletion of other COPI subunits, COPG1 and COPD, similarly induces STING activation, which suggests that innate immune diseases associated with mutations in other COPI subunit genes may exist.
Finally in this chapter, we show that inflammation in COPA syndrome patient PBMCs and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS-STING pathway as a novel therapeutic approach to treat inflammation associated with COPA syndrome and perhaps other COPI-deficiencies.
In the second and third part of this thesis, we focussed on NLRC4-associated AIDs, which are typically caused by autosomal dominant gain of function mutations in NLR family CARD domain containing 4 (NLRC4) that drive hyperactive inflammasome signalling.
Here, we report the first case of a patient with immune dysregulation carrying a recessive mutation in NLRC4 (c.478G>A, p.A160T). Using historical clinical data, serum cytokine analysis and in vitro stimulation of monocyte-derived macrophages we suggest aberrant inflammasome signalling as underlying cause of the inflammatory episodes experienced by the patient.
In vitro, we assessed the pathogenic potential of this mutation and demonstrate ligand-induced increased ASC specking, pyroptosis and cytokine secretion by NLRC4 A160T. Further, using meta-analysis association data we find that heterozygous allele carriers in the general population are at increased risk of developing ulcerative colitis (UC).
Results from subsequently performed structural and biophysical studies aiming to elucidate the conformational changes associated with this mutation point towards a mechanism of action that only affects NLRC4 A160T in the active conformation. Furthermore, since residue A160 is conserved in mice and most primate NLRC4 proteins, but T160 is encoded in the wildtype sequence of NLRC4 in ungulate and carnivore species, the evolutionary relationship between these species is presented. Finally, we discuss potential mechanisms of actions by which NLRC4 A160T may cause aberrant inflammasome activation and evaluate residue T160 as a potential phosphorylation site.
Together, studies described in this thesis identify a direct target for therapeutic intervention in immune diseases associated with defective COPI transport and shape the current understanding of retrograde trafficking as negative regulatory mechanism in STING signalling. Further, we provide in vitro evidence for the pathogenic potential of the NLRC4 A160T mutation, which was previously unrecognized and may represent a susceptibility locus for UC.
Klassifikation (DDC)
570 Biowissenschaften, Biologie 610 Medizin, GesundheitSteiner, Annemarie: Investigation of novel pathways causing autoinflammatory disease. - Bonn, 2022. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn, Universität Melbourne.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-66401
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-66401
@phdthesis{handle:20.500.11811/9804,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-66401,
author = {{Annemarie Steiner}},
title = {Investigation of novel pathways causing autoinflammatory disease},
school = {{Rheinische Friedrich-Wilhelms-Universität Bonn} and {Universität Melbourne}},
year = 2022,
month = may,
note = {Autoinflammatory diseases (AIDs) are rare pathologies caused by mutations in genes associated with innate immune pathways. Investigating the underlying molecular defects contributes to identification of targeted treatment strategies for patients and steadily extends our knowledge of innate signalling pathways and their regulation. Two different diseases were the focus of this thesis: COPA syndrome and NLRC4-AID.
COPA syndrome is an autosomal dominantly inherited immune disorder, caused by loss of function mutations in the coat protein complex I (COPI) subunit α (COPA), which participates in retrograde vesicular trafficking of proteins from the Golgi apparatus to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency of COPA is associated with ER stress, NF-κB activation and type I interferon (IFNαβ) signalling, although the upstream innate immune sensor that is responsible for this was unknown.
Using a range of biochemical and molecular biological techniques, we established in vitro models that recapitulate unprovoked proinflammatory signalling upon CRISPR/Cas9-mediated deletion of COPA and identify aberrant activation of immune sensor STING, which was also dependent on upstream cytosolic DNA sensor cyclic GMP-AMP-synthase (cGAS).
Further, we demonstrate that genetic deletion of other COPI subunits, COPG1 and COPD, similarly induces STING activation, which suggests that innate immune diseases associated with mutations in other COPI subunit genes may exist.
Finally in this chapter, we show that inflammation in COPA syndrome patient PBMCs and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS-STING pathway as a novel therapeutic approach to treat inflammation associated with COPA syndrome and perhaps other COPI-deficiencies.
In the second and third part of this thesis, we focussed on NLRC4-associated AIDs, which are typically caused by autosomal dominant gain of function mutations in NLR family CARD domain containing 4 (NLRC4) that drive hyperactive inflammasome signalling.
Here, we report the first case of a patient with immune dysregulation carrying a recessive mutation in NLRC4 (c.478G>A, p.A160T). Using historical clinical data, serum cytokine analysis and in vitro stimulation of monocyte-derived macrophages we suggest aberrant inflammasome signalling as underlying cause of the inflammatory episodes experienced by the patient.
In vitro, we assessed the pathogenic potential of this mutation and demonstrate ligand-induced increased ASC specking, pyroptosis and cytokine secretion by NLRC4 A160T. Further, using meta-analysis association data we find that heterozygous allele carriers in the general population are at increased risk of developing ulcerative colitis (UC).
Results from subsequently performed structural and biophysical studies aiming to elucidate the conformational changes associated with this mutation point towards a mechanism of action that only affects NLRC4 A160T in the active conformation. Furthermore, since residue A160 is conserved in mice and most primate NLRC4 proteins, but T160 is encoded in the wildtype sequence of NLRC4 in ungulate and carnivore species, the evolutionary relationship between these species is presented. Finally, we discuss potential mechanisms of actions by which NLRC4 A160T may cause aberrant inflammasome activation and evaluate residue T160 as a potential phosphorylation site.
Together, studies described in this thesis identify a direct target for therapeutic intervention in immune diseases associated with defective COPI transport and shape the current understanding of retrograde trafficking as negative regulatory mechanism in STING signalling. Further, we provide in vitro evidence for the pathogenic potential of the NLRC4 A160T mutation, which was previously unrecognized and may represent a susceptibility locus for UC.},
url = {https://hdl.handle.net/20.500.11811/9804}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-66401,
author = {{Annemarie Steiner}},
title = {Investigation of novel pathways causing autoinflammatory disease},
school = {{Rheinische Friedrich-Wilhelms-Universität Bonn} and {Universität Melbourne}},
year = 2022,
month = may,
note = {Autoinflammatory diseases (AIDs) are rare pathologies caused by mutations in genes associated with innate immune pathways. Investigating the underlying molecular defects contributes to identification of targeted treatment strategies for patients and steadily extends our knowledge of innate signalling pathways and their regulation. Two different diseases were the focus of this thesis: COPA syndrome and NLRC4-AID.
COPA syndrome is an autosomal dominantly inherited immune disorder, caused by loss of function mutations in the coat protein complex I (COPI) subunit α (COPA), which participates in retrograde vesicular trafficking of proteins from the Golgi apparatus to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency of COPA is associated with ER stress, NF-κB activation and type I interferon (IFNαβ) signalling, although the upstream innate immune sensor that is responsible for this was unknown.
Using a range of biochemical and molecular biological techniques, we established in vitro models that recapitulate unprovoked proinflammatory signalling upon CRISPR/Cas9-mediated deletion of COPA and identify aberrant activation of immune sensor STING, which was also dependent on upstream cytosolic DNA sensor cyclic GMP-AMP-synthase (cGAS).
Further, we demonstrate that genetic deletion of other COPI subunits, COPG1 and COPD, similarly induces STING activation, which suggests that innate immune diseases associated with mutations in other COPI subunit genes may exist.
Finally in this chapter, we show that inflammation in COPA syndrome patient PBMCs and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS-STING pathway as a novel therapeutic approach to treat inflammation associated with COPA syndrome and perhaps other COPI-deficiencies.
In the second and third part of this thesis, we focussed on NLRC4-associated AIDs, which are typically caused by autosomal dominant gain of function mutations in NLR family CARD domain containing 4 (NLRC4) that drive hyperactive inflammasome signalling.
Here, we report the first case of a patient with immune dysregulation carrying a recessive mutation in NLRC4 (c.478G>A, p.A160T). Using historical clinical data, serum cytokine analysis and in vitro stimulation of monocyte-derived macrophages we suggest aberrant inflammasome signalling as underlying cause of the inflammatory episodes experienced by the patient.
In vitro, we assessed the pathogenic potential of this mutation and demonstrate ligand-induced increased ASC specking, pyroptosis and cytokine secretion by NLRC4 A160T. Further, using meta-analysis association data we find that heterozygous allele carriers in the general population are at increased risk of developing ulcerative colitis (UC).
Results from subsequently performed structural and biophysical studies aiming to elucidate the conformational changes associated with this mutation point towards a mechanism of action that only affects NLRC4 A160T in the active conformation. Furthermore, since residue A160 is conserved in mice and most primate NLRC4 proteins, but T160 is encoded in the wildtype sequence of NLRC4 in ungulate and carnivore species, the evolutionary relationship between these species is presented. Finally, we discuss potential mechanisms of actions by which NLRC4 A160T may cause aberrant inflammasome activation and evaluate residue T160 as a potential phosphorylation site.
Together, studies described in this thesis identify a direct target for therapeutic intervention in immune diseases associated with defective COPI transport and shape the current understanding of retrograde trafficking as negative regulatory mechanism in STING signalling. Further, we provide in vitro evidence for the pathogenic potential of the NLRC4 A160T mutation, which was previously unrecognized and may represent a susceptibility locus for UC.},
url = {https://hdl.handle.net/20.500.11811/9804}
}
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